The terminal three enzymes of the heme biosynthetic pathway, coproporphyrinogen oxidase (CPO), protoporphyrinogen oxidase (PPO), and ferrochelatase, catalyze the conversion of coproporphyrinogen to protoporphyrin followed by insertion of ferrous iron to form the end product protoheme. These enzymes are of general biochemical interest not only for their unique catalytic functions, but also because of their vectorial organization across the membrane. Medically they are of importance because decreased activities of any one results in a disease condition known generally as porphyria. The research aims of this proposal are to investigate structure-function relationships of the terminal three enzymes to gain a better understanding of how each one carries out its catalytic role and how naturally occurring mutations of various porphyrias affect the normal functioning of the target enzyme. In addition, the applicants hope to determine the potential catalytic roles of various metal ions and cofactors that are associated with each of these enzymes (e.g., iron in CPO, flavin in PPO, and a [2Fe.2S] cluster in ferrochelatase). The applicants also intend to extend their studies on the in situ membrane organization of these three enzymes and to attempt to determine if additional proteins are involved in this portion of the pathway. The general approach will be to take advantage of the experimental versatility offered by using recombinant enzymes expressed in E. coli. It is hoped that molecular biological approaches in conjunction with biophysical approaches will yield significant new data on the catalytic functioning of each of these enzymes.